The overall objectives of the project is the elucidation of the role of adenin nucleotides in general and diadenosine 5', 5'''-P1, P4-tetraphosphate (Ap4A) in particular, in signalling or controlling the onset of DNA synthesis (entry into S-phase of the cell cycle) in mammalian cells. This event is controlled by cytoplasmic factors acting as positive regulatory signals. I have proposed that adenine nucleotides in the form of compartmentalized ATP acting as a negative regulatory signal and diadenosine 5'- 5'''-P1, P4-tetraphosphate (Ap4A), which is formed from ATP in the back reaction of the amino acid activation step and acts as a positive regulatory signal, can control initiation of DNA synthesis in mammalian cells. Recently it has been reported elswhere that the addition of Ap4A to permeabilized G1-arrested baby hamster kidney cells led to initiation of DNA replication in these cells. Goals set for the coming year include the elucidation of the mechanism by which high ATP levels (above 5 mM) or high ATP/ADP ratios (achieved by the use of an ATP regenerating system in the (3H)dTTP incorporation system) inhibit DNA synthesis in isolated 3T6 (mouse fibroblasts) nuclei. Diadenosine 5', 5''',-P1, P4-tetraphosphate (A4A) (at a concentration of 0.1 mM) was shown to stimulate (3H)dTTP incorporation in isolated 3T6 nuclei from cells which were synchronized in mid or late G1 phase of their cycle. I plan to identify the nature of the ATP inhibition and Ap4A stimulation (initiation of new sites of DNA synthesis or elongation of existing sites) by utilization of well documented techniques.